Method for zymotic production of fine chemicals (mety) containing sulphur

ABSTRACT

The invention relates to methods for the fermentative production of sulfur-containing fine chemicals, in particular L-methionine, by using bacteria which express a nucleotide sequence coding for a methionine synthase (metH) gene.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2003/009453 filed Aug. 26, 2003, which claims benefit of Germanapplication 102 39 082.7 filed Aug. 26, 2002.

DESCRIPTION

The invention relates to a method for the fermentative production ofsulfur-containing fine chemicals, in particular L-methionine, by usingbacteria which express a nucleotide sequence coding for anO-acetylhomoserine sulfhydrolase (metY) gene.

PRIOR ART

Sulfur-containing fine chemicals such as, for example, methionine,homocysteine, S-adenosylmethionine, glutathione, cysteine, biotin,thiamine, lipoic acid are produced in cells via natural metabolicprocesses and are used in many branches of industry, including the food,animal feed, cosmetics and pharmaceutical industries. These substanceswhich are collectively referred to “sulfur-containing fine chemicals”include organic acids, both proteinogenic and nonproteinogenic aminoacids, vitamins and cofactors. They are most expediently produced on alarge scale by means of cultivating bacteria which have been developedin order to produce and secrete large amounts of the substance desiredin each case. Organisms which are particularly suitable for this purposeare coryneform bacteria, Gram-positive nonpathogenic bacteria.

It is known that amino acids are produced by fermentation of strains ofcoryneform bacteria, in particular Corynebacterium glutamicum. Due tothe great importance, the production processes are constantly improved.Process improvements can relate to measures regarding technical aspectsof the fermentation, such as, for example, stirring and oxygen supply,or to the nutrient media composition such as, for example, sugarconcentration during fermentation or to the work-up to give the product,for example by ion exchange chromatography, or to the intrinsicperformance properties of the microorganism itself.

A number of mutant strains which produce an assortment of desirablecompounds from the group of sulfur-containing fine chemicals have beendeveloped via strain selection. The performance properties of saidmicroorganisms are improved with respect to the production of aparticular molecule by applying methods of mutagenesis, selection andmutant selection. However, this is a time-consuming and difficultprocess. In this way strains are obtained, for example, which areresistant to antimetabolites such as, for example, the methionineanalogs α-methylmethionine, ethionine, norleucine, n-acetylnorleucine,S-trifluoromethylhomocysteine, 2-amino-5-heprenoitic acid,selenomethionine, methioninesulfoximine, methoxine,1-aminocyclopentanecarboxylic acid or which are auxotrophic formetabolites important for regulation and which produce sulfur-containingfine chemicals such as, for example, L-methionine.

Methods of recombinant DNA technology have also been used for some yearsto improve Corynebacterium strains producing L-amino acids by amplifyingindividual amino-acid biosynthesis genes and investigating the effect onamino acid production.

WO-A-02/18613 describes the nucleic acid sequence and the amino acidsequence for metY from C. glutamicum and its use for the production ofL-lysine.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a novel method forthe improved fermentative production of sulfur-containing finechemicals, in particular L-methionine.

We have found that this object is achieved by providing a method for thefermentative production of a sulfur-containing fine chemical, comprisingthe expression of a heterologous nucleotide sequence coding for aprotein with metY activity in a coryneform bacterium.

The invention firstly relates to a method for the fermentativeproduction of at least one sulfur-containing fine chemical, whichcomprises the following steps:

-   a) fermentation of a coryneform bacteria culture producing the    desired sulfur-containing fine chemical, the coryneform bacteria    expressing at least one heterologous nucleotide sequence which codes    for a protein with O-acetylhomoserine sulfhydrolase (metY) activity;-   b) concentration of the sulfur-containing fine chemical in the    medium or in the bacterial cells, and-   c) isolation of the sulfur-containing fine chemical, which    preferably comprises L-methionine.

The above heterologous metY-encoding nucleotide sequence is preferablyless than 100%, such as, for example, more than 70%, such as 75, 80, 85,90 or 95%, or less than 70%, such as, for example, up to 60, 50, 40, 30,20 or 10% homologous to the metY-encoding sequence from Corynebacteriumglutamicum ATCC 13032. The metY-encoding sequence is derived preferablyfrom any of the following organisms of list I:

List I Corynebacterium diphteriae ATCC 14779 Mycobacterium tuberculosisATCC 25584 CDC1551 Clostridium acetobutylicum ATCC 824 Bacillushalodurans ATCC21591 Bacillus stearothermophilus ATCC 12980 Chlorobiumtepidum ATCC 49652 Synechococcus sp. ATCC27104 Emericella nidulans ATCC36104 Bacteroides fragilis ATCC 25285 Lactococcus lactis ATCC 7962Bordetella bronchiseptica ATCC 19395 Pseudomonas aeruginosa ATCC 17933Nitrosomonas europaea ATCC 19718 Sinorhizobium meliloti ATCC 4399Thermotoga maritima ATCC 43589 Streptococcus mutans ATCC 25175Burkholderia cepacia ATCC 25416 Deinococcus radiodurans ATCC 13939Rhodobacter capsulatus ATCC 11166 Pasteurella multocida ATCC 6530Clostridium difficile ATCC 9689 Campylobacter jejuni ATCC 33560Streptococcus pneumoniae ATCC 6308 Saccharomyces cerevisiae ATCC 2704Kluyveromyces lactis ATCC 8585 Candida albicans ATCC 10231Schizosaccharomyces pombe ATCC 24969 ATCC: American Type CultureCollection, Rockville, MD, USA

The metY-encoding sequence used according to the invention preferablycomprises a coding sequence according to SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51 and 53 or a nucleotide sequence homologous thereto which codesfor a protein with metY activity.

Moreover, the metY-encoding sequence used according to the inventionpreferably codes for a protein with metY activity, said proteincomprising an amino acid sequence according to SEQ ID NO:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52 and 54 or an amino acid sequence homologous thereto whichrepresents a protein with metY activity.

The coding metY sequence is preferably a DNA or an RNA which can bereplicated in coryneform bacteria or is stably integrated into thechromosome.

According to a preferred embodiment, the method of the invention iscarried out by

a) using a bacterial strain transformed with a plasmid vector whichcarries at least one copy of the coding metY sequence under the controlof regulatory sequences or

b) using a strain in which the coding metY sequence has been integratedinto the bacterial chromosome.

Furthermore, preference is given to overexpressing the coding metYsequence for the fermentation.

It may also be desirable to ferment bacteria in which additionally atleast one further gene of the biosynthetic pathway of the desiredsulfur-containing fine chemical has been amplified; and/or in which atleast one metabolic pathway, which reduces production of the desiredsulfur-containing fine chemical has, at least partially, been switchedoff.

It may also be desirable to ferment bacteria in which additionally theactivity of at least one further gene of the biosynthetic pathway of thedesired sulfur-containing fine chemical is not undesirably influenced bymetabolic metabolites.

Therefore, according to another embodiment of the method of theinvention, coryneform bacteria are fermented in which, at the same time,at least one of the genes selected from among

-   -   a) the gene lysC, which encodes an aspartate kinase,    -   b) the gene asd, which encodes an aspartate-semialdehyde        dehydrogenase,    -   c) the glyceraldehyde-3-phosphate dehydrogenase-encoding gene        gap,    -   d) the 3-phosphoglycerate kinase-encoding gene pgk,    -   e) the pyruvate carboxylase-encoding gene pyc,    -   f) the triose phosphate isomerase-encoding gene tpi,    -   g) the homoserine O-acetyltransferase-encoding gene meta,    -   h) the cystathionine gamma-synthase-encoding gene metB,    -   i) the cystathionine gamma-lyase-encoding gene metC,    -   j) the serine hydroxymethyltransferase-encoding gene glyA,    -   k) the methionine synthase-encoding gene metH,    -   l) the methylene tetrahydrofolate reductase-encoding gene metF,    -   m) the phosphoserine aminotransferase-encoding gene serC,    -   n) the phosphoserine phosphatase-encoding gene serB,    -   o) the serine acetyl transferase-encoding gene cysE,    -   p) the homoserine dehydrogenase-encoding gene hom is        overexpressed.

According to another embodiment of the method of the invention,coryneform bacteria are fermented in which, at the same time, at leastone of the genes selected from among genes of the abovementioned groupa) to p) is mutated in such a way that the activity of the correspondingproteins is influenced by metabolic metabolites to a smaller extent, ifat all, compared to nonmutated proteins and that in particular theinventive production of the fine chemical is not adversely affected.

According to another embodiment of the method of the invention,coryneform bacteria are fermented in which, at the same time, at leastone of the genes selected from among

-   -   q) the homoserine kinase-encoding gene thrB,    -   r) the threonine dehydratase-encoding gene ilvA,    -   s) the threonine synthase-encoding gene thrC,    -   t) the meso-diaminopimelate D-dehydrogenase-encoding gene ddh,    -   u) the phosphoenolpyruvate carboxykinase-encoding gene pck,    -   v) the glucose-6-phosphate 6-isomerase-encoding gene pgi,    -   w) the pyruvate oxidase-encoding gene poxB,    -   x) the dihydrodipicolinate synthase-encoding gene dapA,    -   y) the dihydrodipicolinate reductase-encoding gene dapB; or    -   z) the diaminopicolinate decarboxylase-encoding gene lysA is        attenuated, in particular by reducing the rate of expression of        the corresponding gene.

According to another embodiment of the method of the invention,coryneform bacteria are fermented in which, at the same time, at leastone of the genes of the above groups q) to z) is mutated in such a waythat the enzymic activity of the corresponding protein is partially orcompletely reduced.

Preference is given to using, in the method of the invention,microorganisms of the species Corynebacterium glutamicum.

The invention further relates to a method for producing anL-methionine-containing animal feed additive from fermentation broths,which comprises the following steps:

-   -   a) culturing and fermentation of an L-methionine-producing        microorganism in a fermentation medium;    -   b) removal of water from the L-methionine-containing        fermentation broth;    -   c) removal of from 0 to 100% by weight of the biomass formed        during fermentation; and    -   d) drying of the fermentation broth obtained according to b)        and/or c), in order to obtain the animal feed additive in the        desired powder or granule form.

The invention likewise relates to the coding metY sequences isolatedfrom the above microorganisms for the first time, to theO-acetylhomoserine sulfhydrolases encoded thereby and to the functionalhomologs of these polynucleotides and proteins, respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the plasmid map for plasmid pC lysC.

FIG. 2 shows the plasmid map for plasmid pCIS lysC thr311lile.

FIG. 3 shows the plasmid map for plasmid pG Phsdh metY_Mt (Mycobacteriumtuberculosis).

DETAILED DESCRIPTION OF THE INVENTION

a) General Terms

Proteins with O-acetylhomoserine sulfhydrolase activity, also referredto as metY (EC 4.2.99.10), are described as being proteins which arecapable of converting O-acetylhomoserine and sulfide into homocystein,using the cofactor pyrodoxal phosphate. The skilled worker distinguishesbetween the activity of O-acetylhomoserine sulfhydrolase and that ofO-succinylhomoserine sulfhydrolase, also referred to as metz. In thelatter enzyme, O-succinylhomoserine and not O-acetylhomoserine acts asthe substrate for the reaction. The skilled worker can detect theenzymatic activity of mety by means of enzyme assays, protocols forwhich may be: Shimizu H. Yamagata S. Masui R. Inoue Y. Shibata T.Yokoyama S. Kuramitsu S. Iwama T. Biochimica et Biophysica Acta.1549(1):61-72, 2001, Yamagata S. Isaji M. Nakamura K. Fujisaki S. Doi K.Bawden S. D'Andrea R. Applied Microbiology & Biotechnology. 42(1):92-9,1994.

Within the scope of the present invention, the term “sulfur-containingfine chemical” includes any chemical compound which contains at leastone covalently bound sulfur atom and is accessible by a fermentationmethod of the invention. Nonlimiting examples thereof are methionine,homocysteine, S-adenosylmethionine, in particular methionine andS-adenosylmethionine.

Within the scope of the present invention, the terms “L-methionine”,“methionine”, homocysteine and S-adenosylmethionine also include thecorresponding salts such as, for example, methionine hydrochloride ormethionine sulfate.

“Polynucleotides” in general refers to polyribonucleotides (RNA) andpolydeoxynbonucleotides (DNA) which may be unmodified RNA and DNArespectively, or modified RNA and DNA, respectively.

According to the invention, “polypeptides” means peptides or proteinswhich contain two or more amino acids linked via peptide bonds.

The term “metabolic metabolite” refers to chemical compounds which occurin the metabolism of organisms as intermediates or else final productsand which, apart from their property as chemical building blocks, mayalso have a modulating effect on enzymes and on their catalyticactivity. It is known from the literature that such metabolicmetabolites may act on the activity of enzymes in both an inhibiting anda stimulating manner (Biochemistry, Stryer, Lubert, 1995 W. H. Freeman &Company, New York, N.Y.). The possibility of producing in organismsenzymes in which the influence of metabolic metabolites has beenmodified by measures such as mutation of the genomic DNA by UVradiation, ionizing radiation or mutagenic substances and subsequentselection for particular phenotypes has also been described in theliterature (Sahm H., Eggeling L., de Graaf A A., Biological Chemistry381(9-10):899-910, 2000; Eikmanns B J., Eggeling L., Sahm H., Antonievan Leeuwenhoek., 64:145-63, 1993-94). These altered properties may alsobe achieved by specific measurements. The skilled worker knows that itis possible specifically to modify in enzyme genes particularnucleotides of the DNA coding for the protein that the protein resultingfrom the expressed DNA sequence has certain new properties, in such away for example that the modulating effect of metabolic metabolites onthe unmodified protein has changed.

The activity of enzymes may be influenced in such a way that thereaction rate is reduced or the affinity for the substrate is modifiedor the reaction rates are changed.

The terms “express” and “amplification” or “overexpression” describe inthe context of the invention the production of or increase inintracellular activity of one or more enzymes encoded by thecorresponding DNA in a microorganism. For this purpose, for example, itis possible to introduce a gene into an organism, to replace an existinggene by another gene, to increase the copy number of the gene or genes,to use a strong promoter or to use a gene which codes for acorresponding enzyme having a high activity, and these measures can becombined, where appropriate.

b) MetY Proteins of the Invention

The invention likewise includes “functional equivalents” of thespecifically disclosed metY enzymes of organisms in the above list I.

Within the scope of the present invention, “functional equivalents” oranalogs of the specifically disclosed polypeptides are polypeptidesdifferent therefrom, which furthermore have the desired biologicalactivity such as, for example, substrate specificity.

According to the invention, “functional equivalents” means in particularmutants which have in at least one of the abovementioned sequencepositions an amino acid other than the specifically mentioned aminoacid, but which have nevertheless one of the abovementioned biologicalactivities. “Functional equivalents” thus also include the mutantsobtainable by one or more amino acid additions, substitutions, deletionsand/or inversions, it being possible for said modifications to occur atany position in the sequence as long as they result in a mutant havingthe property profile of the invention. There is functional equivalencein particular also when the reaction patterns of mutant and unmodifiedpolypeptide match qualitatively, i.e. identical substrates are convertedwith different rates, for example.

“Functional equivalents” naturally also comprise polypeptides which areobtainable from other organisms, and naturally occurring variants. Forexample, homologous sequence regions can be found by sequencecomparison, and equivalent enzymes can be established following thespecific guidelines of the invention.

“Functional equivalents” likewise comprise fragments, preferablyindividual domains or sequence motifs, of the polypeptides of theinvention, which have the desired biological function, for example.

“Functional equivalents” are also fusion proteins which have one of theabovementioned polypeptide sequences or functional equivalents derivedtherefrom and at least one further heterologous sequence functionallydifferent therefrom in functional N- or C-terminal linkage (i.e. withnegligible functional impairment of the functions of the fusion proteinparts). Nonlimiting examples of such heterologous sequences are, forexample, signal peptides, enzymes, immunoglobulins, surface antigens,receptors or receptor ligands.

According to the invention, “functional equivalents” include homologs ofthe specifically disclosed proteins. These have at least 30%, or about40%, 50%, preferably at least about 60%, 65%, 70%, or 75%, in particularat least 85%, such as, for example, 90%, 95% or 99%, homology to one ofthe specifically disclosed sequences, calculated by the algorithm ofPearson and Lipman, Proc. Natl. Acad., Sci. (USA) 85(8), 1988,2444-2448.

Homologs of the proteins or polypeptides of the invention can begenerated by mutagenesis, for example by point mutation or truncation ofthe protein. The term “homolog”, as used herein, relates to a variantform of the protein, which acts as agonist or antagonist of the proteinactivity.

Homologs of the proteins of the invention can be identified by screeningcombinatorial libraries of mutants such as, for example, truncationmutants. It is possible, for example, to generate a variegated libraryof protein variants by combinatory mutagenesis at the nucleic acidlevel, for example by enzymatic ligation of a mixture of syntheticoligonucleotides. There is a multiplicity of methods which can be usedfor preparing libraries of potential homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic gene can then be ligated into a suitable expression vector.The use of a degenerate set of genes makes it possible to provide wholesequences which encode the desired set of potential protein sequences inone mixture. Methods for synthesizing degenerate oligonucleotides areknown to the skilled worker (for example, Narang, S. A., (1983)Tetrahedron 39:3; Itakura et al., (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al., (1983) NucleicAcids Res. 11:477).

In addition, libraries of fragments of the protein codon can be used togenerate a variegated population of protein fragments for screening andfor subsequent selection of homologs of a protein of the invention. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double-stranded PCR fragment of a coding sequence with anuclease under conditions under which nicking occurs only about once permolecule, denaturing the double-stranded DNA, renaturing the DNA to formdouble-stranded DNA which may comprise sense/antisense pairs of variousnicked products, removing single-stranded sections from newly formedduplexes by treatment with S1 nuclease and ligating the resultingfragment library into an expression vector. It is possible by thismethod to devise an expression library which encodes N-terminal,C-terminal and internal fragments of the protein of the invention, whichhas different sizes.

Several techniques are known in the prior art for screening geneproducts from combinatorial libraries which have been produced by pointmutations or truncation and for screening cDNA libraries for geneproducts with a selected property. These techniques can be adapted torapid screening of gene libraries which have been generated bycombinatorial mutagenesis of homologs of the invention. The mostfrequently used techniques for screening large gene libraries undergoinghigh-throughput analysis comprise the cloning of the gene library intoreplicable expression vectors, transformation of suitable cells with theresulting vector library and expression of the combinatorial genes underconditions under which detection of the desired activity facilitatesisolation of the vector encoding the gene whose product has beendetected. Recursive ensemble mutagenesis (REM), a technique whichincreases the frequency of functional mutants in the libraries, can beused in combination with the screening tests in order to identifyhomologs (Arkin und Yourvan (1992) PNAS 89:7811-7815; Delgrave et al.(1993) Protein Engineering 6(3):327-331

c) Polynucleotides of the Invention

The invention also relates to nucleic acid sequences (single- anddouble-stranded DNA and RNA sequences such as, for example cDNA andmRNA) coding for one of the above metY enzymes and the functionalequivalents thereof which are obtainable, for example, also by use ofartificial nucleotide analogs.

The invention relates both to isolated nucleic acid molecules which codefor polypeptides or proteins of the invention or for biologically activesections thereof, and to nucleic acid fragments which can be used, forexample, for use as hybridization probes or primers for identifying oramplifying coding nucleic acids of the invention.

Moreover, the nucleic acid molecules of the invention may containuntranslated sequences from the 3′ and/or 5′ ends of the coding regionof the gene.

An “isolated” nucleic acid molecule is separated from other nucleic acidmolecules which are present in the natural source of the nucleic acidand may moreover be essentially free of other cellular material orculture medium if it is prepared by recombinant techniques, or free ofchemical precursors or other chemicals if it is chemically synthesized.

The invention furthermore comprises the nucleic acid moleculescomplementary to the specifically described nucleotide sequences or asection thereof.

The nucleotide sequences of the invention make it possible to generateprobes and primers which can be used for identifying and/or cloninghomologous sequences in other cell types and organisms. Such probes andprimers usually complete a nucleotide sequence region which hybridizesunder stringent conditions to at least about 12, preferably at leastabout 25, such as, for example 40, 50 or 75, consecutive nucleotides ofa sense strand of a nucleic acid sequence of the invention or of acorresponding antisense strand.

Further nucleic acid sequences of the invention are derived from SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51 or 53 and differ therefrom throughaddition, substitution, insertion or deletion of one or morenucleotides, but still code for polypeptides having the desired profileof properties. These may be polynucleotides which are identical to abovesequences in at least about 50%, 55%, 60%, 65%, 70%, 80% or 90%,preferably in at least about 95%, 96%, 97%, 98% or 99%, of the sequencepositions.

The invention also includes those nucleic acid sequences which comprise“silent” mutations or are modified, by comparison with a specificallymentioned sequence, in accordance with the codon usage of a specificsource or host organism, as well as naturally occurring variants suchas, for example, splice variants or allelic variants. The inventionlikewise relates to sequences which are obtainable by conservativenucleotide substitutions (i.e. the relevant amino acid is replaced by anamino acid of the same charge, size, polarity and/or solubility).

The invention also relates to molecules derived from specificallydisclosed nucleic acids through sequence polymorphisms. These geneticpolymorphisms may exist because of the natural variation betweenindividuals within a population. These natural variations usually resultin a variance of from 1 to 5% in the nucleotide sequence of a gene.

The invention furthermore also comprises nucleic acid sequences whichhybridize with or are complementary to the abovementioned codingsequences. These polynucleotides can be found on screening of genomic orcDNA libraries, and where appropriate, be amplified therefrom by meansof PCR using suitable primers, and then, for example, be isolated withsuitable probes. Another possibility is to transform suitablemicroorganisms with polynucleotides or vectors of the invention,multiply the microorganisms and thus the polynucleotides, and thenisolate them. An additional possibility is to synthesize polynucleotidesof the invention by chemical routes.

The property of being able to “hybridize” to polynucleotides means theability of a polynucleotide or oligonucleotide to bind under stringentconditions to an almost complementary sequence, while there are nounspecific bindings between noncomplementary partners under theseconditions. For this purpose, the sequences should be 70-100%,preferably 90-100%, complementary. The property of complementarysequences being able to bind specifically to one another is made use of,for example, in the Northern or Southern blot technique or in PCR orRT-PCR in the case of primer binding. Oligonucleotides with a length of30 base pairs or more are usually employed for this purpose. Stringentconditions means, for example, in the Northern blot technique the use ofa washing solution at 50-70° C., preferably 60-65° C., for example0.1×SSC buffer with 0.1% SDS (20×SSC; 3M NaCl, 0.3M Na citrate, pH 7.0)for eluting nonspecifically hybridized cDNA probes or oligonucleotides.In this case, as mentioned above, only nucleic acids with a high degreeof complementarity remain bound to one another. The setting up ofstringent conditions is known to the skilled worker and is described,for example, in Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

c) Isolation of the Coding MetY Gene

The metY genes coding for the enzyme O-acetylhomoserine sulfhydrolasecan be isolated from the organisms of the above list I in a manner knownper se.

In order to isolate the metY genes or else other genes of the organismsof the above list I, first a gene library of this organism is generatedin Escherichia coli (E. coli). The generation of gene libraries isdescribed in detail in generally known textbooks and manuals. Exampleswhich may be mentioned are the textbook by Winnacker: Gene und Klone,Eine Einfuhrung in die Gentechnologie (Vertag Chemie, Weinheim, Germany,1990), and the manual by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A verywell-known gene library is that of E. coli K-12 strain W3110, which wasgenerated in λ vectors by Kohara et al. (Cell 50, 495-508 (1980).

In order to produce a gene library from organisms of list I in E. coli,cosmids such as the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84: 2160-2164), orelse plasmids such as pBR322 (BoliVal; Life Sciences, 25, 807-818(1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) can be used.Suitable hosts are in particular those E. coli strains which arerestriction and recombination defective. An example of this is thestrain DH5αmcr which has been described by Grant et al. (Proceedings ofthe National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNAfragments cloned with the aid of cosmids may then in turn be subclonedinto common vectors suitable for sequencing and subsequently besequenced, as described, for example, in Sanger et al. (proceedings ofthe National Academy of Sciences of the United States of America, 74:5463-5467, 1977).

The DNA sequences obtained can then be studied using known algorithms orsequence analysis programs such as, for example, those by Staden(Nucleic Acids Research 14, 217-232(1986)), by Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program by Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

The metY-encoding DNA sequences from organisms according to the abovelist I were found. In particular, DNA sequences according to SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51 and 53 were found. Furthermore, the aminoacid sequences of the corresponding proteins were derived from said DNAsequences present, using the above-described methods. SEQ ID NO:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52 and 54 depict the resulting amino acid sequences ofthe metY gene products.

Coding DNA sequences which result from the sequences according to SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51 and 53 due to the degeneracy of thegenetic code are likewise subject of the invention. In the same way, theinvention relates to DNA sequences which hybridize with said sequencesor parts of sequences derived therefrom.

Instructions for identifying DNA sequences by means of hybridization canbe found by the skilled worker, inter alia, in the manual “The DIGSystem Users Guide fur Filter Hybridization” from Boehringer MannheimGmbH (Mannheim, Germany, 1993) and in Liebl et al. (InternationalJournal of Systematic Bacteriology (1991) 41: 255-260). Instructions foramplifying DNA sequences with the aid of the polymerase chain reaction(PCR) can be found by the skilled worker, inter alia, in the manual byGait: Oligonucleotide synthesis: A Practical Approach (IRL Press,Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum AkademischerVerlag, Heidelberg, Germany, 1994).

It is furthermore known that changes at the N- and/or C-terminus of aprotein do not substantially impair its function or may even stabilizesaid function. Information on this can be found by the skilled worker,inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169: 751-757(1987)), in O'Regan et al. (Gene 77: 237-251 (1989), in Sahin-Toth etal. (Protein Sciences 3: 240-247 (1994)), in Hochuli et al.(Biotechnology 6: 1321-1325 (1988)) and in known textbooks of geneticsand molecular biology.

Amino acid sequences which result accordingly from SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52 and 54 are likewise part of the invention.

d) Host Cells Used According to the Invention

The invention further relates to microorganisms serving as host cells,in particular coryneform bacteria, which contain a vector, in particulara shuttle vector or plasmid vector, carrying at least one metY gene asdefined by the invention or in which a metY gene of the invention isexpressed or amplified.

These microorganisms can produce sulfur-containing fine chemicals, inparticular L-methionine, from glucose, sucrose, lactose, fructose,maltose, molasses, starch, cellulose or from glycerol and ethanol. Saidmicroorganisms are preferably coryneform bacteria, in particular of thegenus Corynebacterium. Of the genus Corynebacterium, mention must bemade in particular of the species Corynebacterium glutamicum which isknown in the art for its ability to produce L-amino acids.

Examples of suitable strains of coryneform bacteria, which may bementioned, are those of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum (C. glutamicum), such as

Corynebacterium glutamicum ATCC 13032,

Corynebacterium acetoglutamicum ATCC 15806,

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium thermoaminogenes FERM BP-1539,

Corynebacterium melassecola ATCC 17965 or

of the genus Brevibacterium, such as

Brevibacterium flavum ATCC 14067

Brevibacterium lactofermentum ATCC 13869 and

Brevibacterium divaricatum ATCC 14020;

Or strains derived therefrom such as

Corynebacterium glutamicum KFCC10065

Corynebacterium glutamicum ATCC21608

which likewise produce the desired fine chemical or the precursor(s)thereof.

The abbreviation KFCC means the Korean Federation of Culture Collection,the abbreviation ATCC means the American Type Strain Culture Collection,and the abbreviation FERM means the collection of the National Instituteof Bioscience and Human Technology, Agency of Industrial Science andTechnology, Japan.

e) Carrying out the Fermentation of the Invention

According to the invention, it was found that coryneform bacteria, afteroverexpression of a metY gene from organisms of the list I, producesulfur-containing fine chemicals, in particular L-methionine, in anadvantageous manner.

To achieve overexpression, the skilled worker can take differentmeasures individually or in combination. Thus it is possible to increasethe copy number of the appropriate genes or to mutate the promoter andregulatory region or the ribosomal binding site which is locatedupstream of the structural gene. Expression cassettes which areincorporated upstream of the structural gene act in the same way.Inducible promoters make it additionally possible to increase expressionduring the course of the fermentative L-methionine production.Expression is likewise improved by measures which extend the life spanof the mRNA. Furthermore, the enzymic activity is likewise enhanced bypreventing degradation of the enzyme protein. The genes or geneconstructs may be either present in plasmids with varying copy number orintegrated and amplified in the chromosome. A further possiblealternative is to achieve overexpression of the relevant genes bychanging the media composition and management of the culture.Instructions for this can be found by the skilled worker, inter alia, inMartin et al. (Biontechnology 5, 137-146 (1987)), in Guerrero et al.(Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6,428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in theEuropean patent 0472869, in U.S. Pat. No. 4,601,893, in Schwarzer andPühler (Biotechnology 9, 84-87 (1991), in Remscheid et al. (Applied andEnvironmental Microbiology 60, 126-132 (1994), in LaBarre et al.(Journal of Bacteriology 175, 1001-1007 (1993)), in the patentapplication WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)),in the Japanese published specification JP-A-10-229891, in Jensen undHammer (Biotechnology and Bioengineering 58,191-195 (1998)), in Makrides(Microbiological Reviews 60: 512-538 (1996) and in known textbooks ofgenetics and molecular biology.

The invention therefore also relates to expression constructs comprisinga nucleic acid sequence coding for a polypeptide of the invention underthe genetic control of regulatory nucleic acid sequences; and to vectorscomprising at least one of said expression constructs. Such constructsof the invention preferably include a promoter 5′ upstream of theparticular coding sequence and a terminator sequence 3′ downstream andalso, where appropriate, further regulatory elements, in each caseoperatively linked to the coding sequence. An “operative linkage” meansthe sequential arrangement of promoter, coding sequence, terminator and,where appropriate, further regulatory elements such that each of theregulatory elements can properly carry out its function in theexpression of the coding sequence. Examples of operatively linkablesequences are activating sequences and enhancers and the like. Furtherregulatory elements include selectable markers, amplification signals,origins of replication and the like. Suitable regulatory sequences aredescribed, for example in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990).

In addition to the artificial regulatory sequences, the naturalregulatory sequence may still be present upstream of the actualstructural gene. Genetic modification can, where appropriate, switch offthis natural regulation and increase or decrease expression of thegenes. However, the gene construct may also have a simpler design, i.e.no additional regulatory signals are inserted upstream of the structuralgene and the natural promoter with its regulation is not removed.Instead, the natural regulatory sequence is mutated such that regulationno longer takes place and gene expression is increased or reduced. Thegene construct may contain one or more copies of the nucleic acidsequences.

Examples of useful promoters are: ddh, amy, lysC, dapA, lysA fromCorynebacterium glutamicum promoters, but also Gram-positive promotersSPO02, as are described in Bacillus Subtilis and Its Closest Relatives,Sonenshein, Abraham L., Hoch, James A., Losick, Richard; ASM Press,District of Columbia, Washington and Patek M. Eikmanns B J., Patek J.,Sahm H., Microbiology. 142 1297-309, 1996 or else the cos, tac, trp,tet, trp-tet, Ipp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6,λ-PR and λ-PL promoters which are advantageously applied inGram-negative bacteria. Preference is also give to using induciblepromoters such as, for example light- and, in particular,temperature-inducible promoters such as the P_(r)P_(l) promoter. It isin principle possible to use all natural promoters with their regulatorysequences. In addition, it is also possible to use advantageouslysynthetic promoters.

The regulatory sequences mentioned are intended to make specificexpression of the nucleic acid sequences possible. Depending on the hostorganism, this may mean, for example, that the gene is expressed oroverexpressed only after induction, or that it is immediately expressedand/or overexpressed.

In this connection, the regulatory sequences and factors may preferablyhave a beneficial effect on, and thus increase or decrease, expression.Thus, it is possible and advantageous to enhance the regulatory elementsat the transcriptional level by using strong transcription signals suchas promoters and/or enhancers. However, it is also possible besides thisto enhance translation by, for example, improving the stability of themRNA.

An expression cassette is prepared by fusing a suitable promoter, asuitable Shine-Dalgarno sequence, to a metY nucleotide sequence and asuitable termination signal. For this purpose, common recombination andcloning techniques are used, such as those described, for example, inCurrent Protocols in Molecular Biology, 1993, John Wiley & Sons,Incorporated, New York, New York, PCR Methods, Gelfand, David H., Innis,Michael A., Sninsky, John J., 1999, Academic Press, Incorporated,California, San Diego, PCR Cloning Protocols, Methods in MolecularBiology Ser., Vol. 192, 2nd ed., Humana Press, New Jersey, Totowa. T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)and in T. J. Silhavy, M. L. Berman und L. W. Enquist, Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1984) and in Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley Interscience (1987).

The recombinant nucleic acid construct or gene construct is expressed ina suitable host organism by inserting it advantageously into ahost-specific vector which makes optimal expression of the genes in thehost possible. Vectors are well known to the skilled worker and can befound, for example, in “Cloning Vectors” (Pouwels P. H. et al., Hrsg,Elsevier, Amsterdam-New York-Oxford, 1985). The term “vectors” means,apart from plasmids, also all other vectors known to the skilled worker,such as, for example, phages, transposons, IS elements, plasmids,cosmids and linear or circular DNA. These vectors can replicateautonomously in the host organism or are replicated chromosomally.

MetY genes of the invention were amplified by overexpressing them by wayof example with the aid of episomal plasmids. Suitable plasmids arethose which are replicated in coryneform bacteria. Numerous knownplasmid vectors such as, for example, pZ1 (Menkel et al., Applied andEnvironmental Microbiology (1989) 64: 549-554), pEKE×1 (Eikmanns et al.,Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74(1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Otherplasmid vectors such as, for example, pCLiK5MCS, or those based on pCG4(U.S. Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMSMicrobiology Letters 66, 119-124 (1990)) or pAG1 (U.S. Pat. No.5,158,891) may be used in the same way.

Suitable plasmid vectors are furthermore also those with the aid ofwhich it is possible to apply the method of gene amplification byintegration into the chromosome, as has been described, for example, byRemscheid et al. (Applied and Environmental Microbiology 60,126-132(1994)) for the duplication and amplification of the hom-thrB operon. Inthis method, the complete gene is cloned into a plasmid vector which canreplicate in a host (typically E. coli) but not in C. glutamicum.Suitable vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schafer et al., Gene 145, 69-73(1994)), Bernard et al., Journal of Molecular Biology, 234: 534-541(1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342). The plasmidvector containing the gene to be amplified is then transferred into thedesired C. glutamicum strain via transformation. Methods fortransformation are described, for example, in Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Biotechnology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)).

The activity of enzymes can be influenced by mutations in thecorresponding genes in such a way that the rate of the enzymic reactionis partly or completely reduced. Examples of such mutations are known tothe skilled worker (Motoyama H., Yano H., Terasaki Y., Anazawa H.,Applied & Environmental Microbiology. 67:3064-70, 2001, Eikmanns B J.,Eggeling L., Sahm H., Antonie van Leeuwenhoek. 64:145-63, 1993-94.)

Additionally, it may be advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, toamplify, in addition to expression and amplification of a metY gene ofthe invention, one or more enzymes of the respective biosyntheticpathway, the cysteine pathway, of aspartate-semialdehyde synthesis, ofglycolysis, of anaplerosis, of the pentose phosphate metabolism, thecitrate acid cycle or the amino acid export.

Thus, one or more of the following genes can be amplified to producesulfur-containing fine chemicals, in particular L-methionine:

-   -   the gene lysC, which encodes an aspartate kinase (EP 1 108 790        A2; DNA-SEQ NO. 281),    -   the gene asd which encodes an aspartate-semialdehyde        dehydrogenase (EP 1 108 790 A2; DNA-SEQ NO. 282),    -   the glyceraldehyde-3-phosphate dehydrc ogenase-encoding gene gap        (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),    -   the 3-phosphoglycerate kinase-encoding gene pgk (Eikmanns        (1992), Journal of Bacteriology 174: 6076-6086),    -   the pyruvate carboxylase-encoding gene pyc (Eikmanns (1992),        Journal of Bacteriology 174: 6076-6086),    -   the triose phosphate isomerase-encoding gene tpi (Eikmanns        (1992), Journal of Bacteriology 174: 6076-6086),    -   the homoserine O-acetyltransferase-encoding gene metA (EP 1 108        790 A2; DNA-SEQ NO. 725),    -   the cystathionine gamma-synthase-encoding gene metB (EP 1 108        790 A2; DNA-SEQ NO. 3491),    -   the cystathionine gamma-lyase-encoding gene metC (EP 1 108 790        A2; DNA-SEQ NO. 3061),    -   the serine hydroxymethyltransferase-encoding gene glyA (EP 1 108        790 A2; DNA-SEQ NO. 1110),    -   the methionine synthase-encoding gene metH (EP 1 108 790 A2),    -   the methylene tetrahydrofolate reductase-encoding gene metF (EP        1 108 790 A2; DNA-SEQ NO. 2379),    -   the phosphoserine aminotransferase-encoding gene serC (EP 1 108        790 A2; DNA-SEQ NO. 928),    -   a phosphoserine phosphatase-encoding gene serB (EP 1 108 790 A2;        DNA-SEQ NO. 334, DNA-SEQ NO. 467, DNA-SEQ NO. 2767),    -   the gene cysE, which encodes a serine acetyl transferase (EP 1        108 790 A2; DNA-SEQ NO. 2818),    -   the gene hom, which encodes a homoserine dehydrogenase (EP 1 108        790 A2; DNA-SEQ NO. 1306)

Thus, it may be advantageous for the production of sulfur-containingfine chemicals, in particular L-methionine, in coryneform bacteria tomutate, at the same time, at least one of the genes below, so that theactivity of the corresponding proteins, compared to that of unmutatedproteins, is influenced by a metabolic metabolite to a lesser extent ornot at all:

-   -   the gene lysC, which encodes an aspartate kinase (EP 1 108 790        A2; DNA-SEQ NO. 281),    -   the pyruvate carboxylase-encoding gene pyc (Eikmanns (1992),        Journal of Bacteriology 174: 6076-6086),    -   the homoserine O-acetyltransferase-encoding gene metA (EP 1 108        790 A2; DNA-SEQ NO. 725),    -   the cystathionine gamma-synthase-encoding gene metB (EP 1 108        790 A2; DNA-SEQ NO. 3491),    -   the cystathionine gamma-lyase-encoding gene metC (EP 1 108 790        A2; DNA-SEQ NO. 3061),    -   the serine hydroxymethyltransferase-encoding gene glyA (EP 1 108        790 A2; DNA-SEQ NO. 1110),    -   the methionine synthase-encoding gene metH (EP 1 108 790 A2),    -   the methylene tetrahydrofolate reductase-encoding gene metF (EP        1 108 790 A2; DNA-SEQ NO. 2379),    -   the phosphoserine aminotransferase-encoding gene serC (EP 1 108        790 A2; DNA-SEQ NO. 928),    -   a phosphoserine phosphatase-encoding gene serB (EP 1 108 790 A2;        DNA-SEQ NO. 334, DNA-SEQ NO. 467, DNA-SEQ NO. 2767),    -   the serine acetyl transferase-encoding gene cysE (EP 1 108 790        A2; DNA-SEQ NO. 2818),    -   the gene hom, which encodes a homoserine dehydrogenase (EP 1 108        790 A2; DNA-SEQ NO. 1306)

It may be furthermore advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, inaddition to expression and amplification of one of the metY genes of theinvention, to attenuate one or more of the following genes, inparticular to reduce expression thereof, or to switch them off:

-   -   the homoserine kinase-encoding gene thrB (EP 1 108 790 A2;        DNA-SEQ NO. 3453),    -   the threonine dehydratase-encoding gene ilvA (EP 1 108 790 A2;        DNA-SEQ NO. 2328),    -   the threonine synthase-encoding gene thrC (EP 1 108 790 A2;        DNA-SEQ NO. 3486),    -   the meso-diaminopimelate D-dehydrogenase-encoding gene ddh (EP 1        108 790 A2; DNA-SEQ NO. 3494),    -   the phosphoenolpyruvate carboxykinase-encoding gene pck (EP 1        108 790 A2; DNA-SEQ NO. 3157),    -   the glucose-6-phosphate 6-isomerase-encoding gene pgi (EP 1 108        790 A2; DNA-SEQ NO. 950),    -   the pyruvate oxidase-encoding gene poxB (EP 1 108 790 A2;        DNA-SEQ NO. 2873),    -   the dihydrodipicolinate synthase-encoding gene dapA (EP 1 108        790 A2; DNA-SEQ NO. 3476),    -   the dihydrodipicolinate reductase-encoding gene dapB (EP 1 108        790 A2; DNA-SEQ NO. 3477)    -   the diaminopicolinate decarboxylase-encoding gene lysA (EP 1 108        790 A2; DNA-SEQ NO. 3451)

It may be furthermore advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, inaddition to expression and amplification of one of the metY genes of theinvention in coryneform bacteria, to mutate, at the same time, at leastone of the following genes in such a way that the enzymic activity ofthe corresponding protein is partly or completely reduced:

-   -   the homoserine kinase-encoding gene thrB (EP 1 108 790 A2;        DNA-SEQ NO. 3453),    -   the threonine dehydratase-encoding gene ilvA (EP 1 108 790 A2;        DNA-SEQ NO. 2328),    -   the threonine synthase-encoding gene thrC (EP 1 108 790 A2;        DNA-SEQ NO. 3486),    -   the meso-diaminopimelate D-dehydrogenase-encoding gene ddh (EP 1        108 790 A2; DNA-SEQ NO. 3494),    -   the phosphoenolpyruvate carboxykinase-encoding gene pck (EP 1        108 790 A2; DNA-SEQ NO. 3157),    -   the glucose-6-phosphate 6-isomerase-encoding gene pgi (EP 1 108        790 A2; DNA-SEQ NO. 950),    -   the pyruvate oxidase-encoding gene poxB (EP 1 108 790 A2;        DNA-SEQ NO. 2873),    -   the dihydrodipicolinate synthase-encoding gene dapA (EP 1 108        790 A2; DNA-SEQ NO.3476),    -   the dihydrodipicolinate reductase-encoding gene dapB (EP 1 108        790 A2; DNA-SEQ NO. 3477)    -   the diaminopicolinate decarboxylase-encoding gene lysA (EP 1 108        790 A2; DNA-SEQ NO. 3451)

It may be furthermore advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, apart fromexpression and amplification of a metY gene of the invention, toeliminate unwanted secondary reactions (Nakayama: “Breeding of AminoAcid Producing Microorganisms”, in: Overproduction of MicrobialProducts, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK,1982).

The microorganisms produced according to the invention may be culturedcontinuously or batchwise or in a fed batch or repeated fed batchprocess to produce sulfur-containing fine chemicals, in particularL-methionine. An overview of known cultivation methods can be found inthe textbook by Chmiel (Bioprozeβtechnik 1. Einfuhrung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren und periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must satisfy the demands of the particularstrains in a suitable manner. The textbook “Manual of Methods furGeneral Bacteriology” by the American Society for Bacteriology(Washington D.C., USA, 1981) contains descriptions of culture media forvarious microorganisms.

Said media which can be used according to the invention usually compriseone or more carbon sources, nitrogen sources, inorganic salts, vitaminsand/or trace elements.

Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch and cellulose. Sugars may also beadded to the media via complex compounds such as molasses or otherbyproducts of sugar refining. It may also be advantageous to addmixtures of different carbon sources. Other possible carbon sources areoils and fats such as, for example, soybean oil, sunflower oil, peanutoil and coconut fat, fatty acids such as, for example, palmitic acid,stearic acid and linoleic acid, alcohols such as, for example, glycerol,methanol and ethanol and organic acids such as, for example acetic acidand lactic acid.

Nitrogen sources are usually organic or inorganic hydrogen compounds ormaterials containing said compounds. Examples of nitrogen sourcesinclude ammonia gas or ammonium salts such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,nitrates, urea, amino acids and complex nitrogen sources such ascornsteep liquor, soybean flour, soybean protein, yeast extract, meatextract and others. The nitrogen sources may be used singly or asmixture.

Inorganic salt compounds which may be included in the media comprise thechloride, phosphorus or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or elseorganic sulfur compounds such as mercaptans and thiols may be used assources of sulfur for the production of sulfur-containing finechemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used assources of phosphorus.

Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents includedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid.

The fermentation media used according to the invention usually alsocontain other growth factors such as vitamins or growth promoters, whichinclude, for example, biotin, riboflavin, thiamine, folic acid,nicotinic acid, panthothenate and pyridoxine. Growth factors and saltsare frequently derived from complex media components such as yeastextract, molasses, cornsteep liquor and the like. It is moreoverpossible to add suitable precursors to the culture medium. The exactcomposition of the media heavily depends on the particular experimentand is decided upon individually for each specific case. Information onthe optimization of media can be found in the textbook “AppliedMicrobiol. Physiology, A Practical Approach” (Editors. P. M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growthmedia can also be obtained from commercial suppliers, for exampleStandard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by sterile filtration. The components may be sterilizedeither together or, if required, separately. All media components may bepresent at the start of the cultivation or added continuously orbatchwise, as desired.

The culture temperature is normally between 15° C. and 45° C.,preferably at from 25° C. to 40° and may be kept constant or may bealtered during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for cultivation canbe controlled during cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example, fatty acidpolyglycol esters. To maintain the stability of plasmids it is possibleto add to the medium suitable substances having a selective effect, forexample antibiotics. Aerobic conditions are maintained by introducingoxygen or oxygen-containing gas mixtures such as, for example, air intothe culture. The temperature of the culture is normally 20° C. to 45° C.The culture is continued until formation of the desired product is at amaximum. This aim is normally achieved within 10 to 160 hours.

The fermentation broths obtained in this way, in particular thosecontaining L-methionine, usually contain a dry biomass of from 7.5 to25% by weight.

An additional advantage is to carry out the fermentation under sugarlimitation, at least at the end, but in particular over at least 30% ofthe fermentation period. This means that during this time theconcentration of utilizable sugar in the fermentation medium ismaintained at or reduced to ≧0 to 3 g/l.

The fermentation broth is then processed further. The biomass may,according to requirement, be removed completely or partially from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decanting or a combination of these methodsor be left completely in said broth.

Subsequently, the fermentation broth may be thickened or concentratedusing known methods such as, for example, with the aid of a rotaryevaporator, thin film evaporator, falling film evaporator, by reverseosmosis, or by nanofiltration. This concentrated fermentation broth canthen be worked up by freeze drying, spray drying, spray granulation orby other methods.

However, it is also possible to further purify the sulfur-containingfine chemicals, in particular L-methionine. To this end, theproduct-containing broth, after removing the biomass, is subjected to achromatography using a suitable resin, the desired product or thecontaminations being retained completely or partially on thechromatographic resin. These chromatographic steps can be repeated, ifnecessary, using the same or different chromatographic resin. Theskilled worker is familiar with the selection of suitablechromatographic resins and their most effective application. Thepurified product can be concentrated by filtration or ultrafiltrationand stored at a temperature at which the stability of the product isgreatest.

The identity and purity of the isolated compound(s) can be determined bytechniques of the art. These include high performance liquidchromatography (HPLC), spectroscopic methods, staining methods,thin-layer chromatography, NIRS, enzyme assay or microbiological assays.These analytic methods are summarized in: Patek et al. (1994) Appl.Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70.Ulmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH:Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 andpp. 581-587; Michal, G., (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, Volume 17.

The invention is now described in greater detail with reference to thefollowing nonlimiting examples and with reference to the appendedfigures, in which

FIG. 1 shows the plasmid map for plasmid pClysC;

FIG. 2 the plasmid map for plasmid pClSlysCthr311ile;

FIG. 3 the plasmid map for plasmid pCPhsdhmetY_Mt.

Restriction cleavage sites with the corresponding indication of theirposition in brackets are shown in the plasmid maps. Essential sequencesegments are described in bold. KanR means kanamycin resistance gene;ask means aspartate kinase gene.

EXAMPLE 1 Construction of pCLiK5MCS

First, ampicillin resistance and origin of replication of the vectorpBR322 were amplified using the oligonucleotides p1.3 (SEQ ID NO:55) andp2.3 (SEQ ID NO:56) with the aid of the polymerase chain reaction (PCR).

p1.3 (SEQ ID NO:55) 5′-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3′ p2.3 (SEQ ID NO:56)5′-TCTAGACTCGAGCGGCCGCGGCCGGCCTTTAAATTGAAGACGAAAGG GCCTCG-3′

In addition to sequences complementary to pBR322, the oligonucleotidep1.3 (SEQ ID NO:55) contains in 5′-3′ direction the cleavage sites forthe restriction nucleases SmaI, BamHI, NheI and AscI and theoligonucleotide p2.3 (SEQ ID NO:56) contains in 5′-3′ direction thecleavage sites for the restriction endonucleases XbaI, XhoI, NotI andDraI. The PCR reaction was carried out according to a standard methodsuch as that by Innis et al. (PCR Protocols. A Guide to Methods andApplications, Academic Press (1990)) using PfuTurbo polymerase(Stratagene, La Jolla, USA). The DNA fragment obtained of approximately2.1 kb in size was purified using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. The blunt ends of the DNA fragment wereligated to one another using the rapid DNA ligation kit (RocheDiagnostics, Mannheim) according to the manufacturer's instructions andthe ligation mixture was transformed into competent E. coli XL-1Blue(Stratagene, La Jolla, USA) according to standard methods, as describedin Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold SpringHarbor, (1989)). Plasmid-carrying cells were selected for by plating outonto ampicillin (50 μg/ml)-containing LB agar (Lennox, 1955, Virology,1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK1.

Starting from plasmid pWLT1 (Liebl et al., 1992) as template for a PCRreaction, a kanamycin resistance cassette was amplified using theoligonucleotides neo1 (SEQ ID NO:57) and neo2 (SEQ ID NO:58).

neo1 (SEQ ID NO:57) 5′-GAGATCTAGACCCGGGGATCCGCTAGCGGGCTGCTAAAGGAAGCGGA-3′: neo2 (SEQ ID NO:58) 5′-GAGAGGCGCGCCGCTAGCGTGGGCGAAGAACTCCAGCA-3′:

Apart from the sequences complementary to pWLT1, the oligonucleotideneo1 contains in 5′-3′ direction the cleavage sites for the restrictionendonucleases XbaI, SmaI, BamHI, NheI and the oligonucleotide neo2 (SEQID NO:58) contains in 5′-3′ direction the cleavage sites for therestriction endonucleases AscI and NheI. The PCR reaction was carriedout using PfuTurbo polymerase (Stratagene, La Jolla, USA) according to astandard method such as that of Innis et al. (PCR Protocols. A Guide toMethods and Applications, Academic Press (1990)). The DNA fragmentobtained was approximately 1.3 kb in size was purified using theGFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. The DNA fragmentwas cleaved with restriction endonucleases XbaI and AscI (New EnglandBiolabs, Beverly, USA) and, following that, again purified using theGFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. The vectorpCLiK1 was likewise cleaved with the restriction endonucleases XbaI andAscI and dephosphorylated using alkaline phosphatase (Roche Diagnostics,Mannheim) according to the manufacturer's instructions. Afterelectrophoresis in a 0.8% strength agarose gel, the linearized vector(approx. 2.1 kb) was isolated using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. This vector fragment was ligated with thecleaved PCR fragment with the aid of the rapid DNA ligation kit (RocheDiagnostics, Mannheim) according to the manufacturer's instructions andthe ligation mixture was transformed into competent E. coli XL-1Blue(Stratagene, La Jolla, USA) according to standard methods, as describedin Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold SpringHarbor, (1989)). Plasmid-carrying cells were selected for by plating outonto ampicillin (50 μg/ml)- and kanamycin (20 μg/ml)-containing LB agar(Lennox, 1955, Virology, 1: 190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK2.

The vector pCLiK2 was cleaved with the restriction endonuclease DraI(New England Biolabs, Beverly, USA). After electrophoresis in 0.8%strength agarose gel, an approx. 2.3 kb vector fragment was isolatedusing the GFX™PCR, DNA and gel band purification kit (AmershamPharmacia, Freiburg) according to the manufacturer's instructions. Thisvector fragment was religated with the aid of the rapid DNA ligation kit(Roche Diagnostics, Mannheim) according to the manufacturer'sinstructions and the ligation mixture was transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA) according to standard methods,as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual,Cold Spring Harbor, (1989)). Plasmid-carrying cells were selected for byplating out onto kanamycin (20 μg/ml)-containing LB agar (Lennox, 1955,Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK3.

Starting from plasmid pWLQ2 (Liebl et al., 1992) as template for a PCRreaction, the origin of replication pHM1519 was amplified using theoligonucleotides cg1 (SEQ ID NO:59) and cg2 (SEQ ID NO:60).

cg1 (SEQ ID NO:59) 5′-GAGAGGGCGGCCGCGCAAAGTCCCGCTTCGTGAA-3′: cg2 (SEQ IDNO:60) 5′-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3′:

Apart from the sequences complementary to pWLQ2, the oligonucleotidescg1 (SEQ ID NO:59) and cg2 (SEQ ID NO:60) contain cleavage sites for therestriction endonuclease NotI. The PCR reaction was carried out usingPfuTurbo polymerase (Stratagene, La Jolla, USA) according to a standardmethod such as that of Innis et al. (PCR Protocols. A Guide to Methodsand Applications, Academic Press (1990)). The DNA fragment obtained wasapproximately 2.7 kb in size and was purified using the GFX™PCR, DNA andgel band purification kit (Amersham Pharmacia, Freiburg) according tothe manufacturer's instructions. The DNA fragment was cleaved withrestriction endonuclease NotI (New England Biolabs, Beverly, USA) and,following that, again purified using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. The vector pCLiK3 was likewise cleaved withthe restriction endonuclease NotI and dephosphorylated using alkalinephosphatase (Roche Diagnostics, Mannheim) according to themanufacturer's instructions. After electrophoresis in a 0.8% strengthagarose gel, the linearized vector (approx. 2.3 kb) was isolated usingthe GFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. This vectorfragment was ligated with the cleaved PCR fragment with the aid of therapid DNA ligation kit (Roche Diagnostics, Mannheim) according to themanufacturer's instructions and the ligation mixture was transformedinto competent E. coli XL-1Blue (Stratagene, La Jolla, USA) according tostandard methods, as described in Sambrook et al. (Molecular Cloning. ALaboratory Manual, Cold Spring Harbor, (1989)). Plasmid-carrying cellswere selected for by plating out onto kanamycin (20 μg/ml)-containing LBagar (Lennox, 1955, Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK5.

PCLik5 was extended by a multiple cloning site (MCS) by combining thetwo synthetic essentially complementary oligonucleotides HS445 ((SEQ IDNO:61) and HS446 (SEQ ID NO:62)) which contain cleavage sites for therestriction endonucleases SwaI, XhoI, AatI, ApaI, Asp718, MluI, NdeI,SpeI, EcoRV, SalI, ClaI, BamHI, XbaI and SmaI to give a double-strandedDNA fragment by heating them together to 95° C. followed by slowcooling.

HS445 (SEQ ID NO:61) 5′-TCGAATTTAAATCTCGAGAGGCCTGACGTCGGGCCCGGTACCACGCGTCATATGACTAGTTCGGACCTAGGGATATCGTCGACATCGATGCTCTTCTGCGTTAATTAACAATTGGGATCCTCTAGACCCGGGATTTAAAT-3′: HS446 (SEQ ID NO:62)5′-GATCATTTAAATCCCGGGTCTAGAGGATCCCAATTGTTAATTAACGCAGAAGAGCATCGATGTCGACGATATCCCTAGGTCCGAACTAGTCATATGACGCGTGGTACCGGGCCCGACGTCAGGCCTCTCGAGATTTAAAT-3′

The vector pCLiK5 was cleaved with the restriction endonucleases XhoIand BamHI (New England Biolabs, Beverly, USA) and dephosphorylated usingalkaline phosphatase (I (Roche Diagnostics, Mannheim)) according to themanufacturer's instructions. After electrophoresis in a 0.8% strengthagarose gel, the linearized vector (approx. 5.0 kb) was isolated usingthe GFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. This vectorfragment was ligated with the synthetic double-stranded DNA fragmentwith the aid of the rapid DNA ligation kit (Roche Diagnostics, Mannheim)according to the manufacturer's instructions and the ligation mixturewas transformed into competent E. coli XL-1Blue (Stratagene, La Jolla,USA) according to standard methods as described Sambrook et al.(Molecular Cloning. A Laboratory Manual, Cold Spring Harbor (1989)).Plasmid-carrying cells were selected for by plating out onto kanamycin(20 μg/ml)-containing LB agar (Lennox, 1955, Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturersinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK5MCS.

Sequencing reactions were carried out according to Sanger et al. (1977)Proceedings of the National Academy of Sciences USA 74:5463-5467. Thesequencing reactions were fractionated and analyzed by means of ABIPrism 377 (PE Applied Biosystems, Weiterstadt).

The resultant plasmid pCLiK5MCS is listed as SEQ ID NO: 65.

EXAMPLE 2 Construction of pCLiK5MCS Integrativ SacB

Starting from the plasmid pK19mob (Schäfer et al., Gene 145,69-73(1994))as template for a PCR reaction, the Bacillus subtilis sacB gene (codingfor levan sucrase) was amplified using the oligonucleotides BK1732 andBK1733.

BK1732 (SEQ ID NO:63) 5′-GAGAGCGGCCGCCGATCCTTTTTAACCCATCAC-3′: BK1733(SEQ ID NO:64) 5′-AGGAGCGGCCGCCATCGGCATTTTCTTTTGCG-3′:

Apart from the sequences complementary to pEK19mobsac, theoligonucleotides BK1732 and BK1733 contain cleavage sites for therestriction endonuclease NotI. The PCR reaction was carried out usingPfuTurbo polymerase (Stratagene, La Jolla, USA) using a standard methodlike that of Innis et al. (PCR Protocols. A Guide to Methods andApplications, Academic Press (1990)). The DNA fragment obtained ofapproximately 1.9 kb in size was purified using the GFX™PCR, DNA and gelband purification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. The DNA fragment was cleaved with therestriction endonuclease NotI (New England Biolabs, Beverly, USA) and,following that, again purified using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions.

The vector pCLiK5MCS (prepared according to example 1) was likewisecleaved with the restriction endonuclease NotI and dephosphorylatedusing alkali phosphatase (I (Roche Diagnostics, Mannheim)) according tothe manufacturer's instructions. After electrophoresis in a 0.8%strength agarose gel, an approximately 2.4 kb in size vector fragmentwas isolated using the GFX™PCR, DNA and gel band purification kit(Amersham Pharmacia, Freiburg) according to the manufacturer'sinstructions. This vector fragment was ligated with the cleaved PCRfragment with the aid of the rapid DNA ligation kit (Roche Diagnostics,Mannheim) according to the manufacturer's instructions and the ligationmixture was transformed into competent E. coli XL-1Blue (Stratagene, LaJolla, USA) according to standard methods, as described in Sambrook etal. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor,(1989)). Plasmid-carrying cells were selected for by plating out ontokanamycin (20 μg/ml)-containing LB agar (Lennox, 1955, Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK5MCS integrativ sacB.

Sequencing reactions were carried out according to Sanger et al. (1977)Proceedings of the National Academy of Sciences USA 74:5463-5467. Thesequencing reactions were fractionated and analyzed by means of ABIPrism 377 (PE Applied Biosystems, Weiterstadt).

The resultant plasmid pCLiK5MCS integrativ sacB is listed as SEQ ID NO:66.

It is possible to prepare in an analog manner further vectors which aresuitable for the inventive expression or overproduction of metY genes.

EXAMPLE 3 Isolation of the lysC Gene from the C. glutamicum StrainLU1479

In the first step of the strain construction, it is intended to carryout an allelic substitution of the lysC wild-type gene encoding theenzyme aspartate kinase in C. glutamicum ATCC13032, hereinbelow referredto as LU1479. It is intended to carry out a nucleotide substitution inthe LysC gene so that the amino acid lie is substituted for the aminoacid Thr at position 311 in the resulting protein.

Starting from the chromosomal DNA of LU1479 as template for a PCRreaction, an amplification was carried out with the oligonucleotideprimers SEQ ID NO:67 and SEQ ID NO:68 lysC with the aid of the Pfu-TurboPCR system (Stratagene USA) following the manufacture's instructions.Chromosomal DNA from C. glutamicum ATCC 13032 was prepared as describedby Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994)Microbiology 140:1817-1828. The amplified fragment is flanked at its 5′end by a SalI restriction cleavage and at its 3′ end by an MluIrestriction cleavage. Prior to the cloning step, the amplified fragmentwas restricted by these two restriction enzymes and purified usingGFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg).

SEQ ID NO:67 5′-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3′ SEQ ID NO:685′-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3′

The resulting polynucleotide was cloned into pCLIK5 MCS integrativ SacB(hereinbelow referred to as pCIS; SEQ ID NO: 66 of Example 2) via theSalI and MluI restriction cleavages and transformed into E. coli XL-1blue. A selection for plasmid-bearing cells was achieved by plating ontokanamycin (20 μg/ml)-containing LB agar (Lennox, 1955, Virology, 1:190).The plasmid was isolated and the expected nucleotide sequence wasverified by sequencing. The plasmid DNA preparation was carried out bymethods of, and using material from, Quiagen. Sequencing reactions werecarried out as described by Sanger et al. (1977) Proceedings of theNational Academy of Sciences USA 74:5463-5467. The sequencing reactionswere separated by means of ABI Prism 377 (PE Applied Biosystems,Weiterstadt) and evaluated. The resulting plasmid pCIS lysC is shown asSEQ ID NO:69. The corresponding plasmid map is shown in FIG. 1.

The sequence SEQ ID NO:69 encompasses the following essentialpart-regions:

LOCUS pCIS\lysC  5860 bp  DNA  circular FEATURES Location/QualifiersCDS¹⁾   155 . . . 1420 /vntifkey = “4” /label = lysC CDS  complement²⁾(3935 . . . 5356) /vntifkey = “4” /label =sacB\(Bacillus\subtilis) promoter   complement(5357 . . . 5819)/vntifkey = “30” /label = Promotor\sacB C_region   complement(3913 . . .3934) /vntifkey = “2” /label = sacB\downstream region CDS   1974 . . .2765 /vntifkey = “4” /label = Kan\R CDS   complement(3032 . . . 3892)/vntifkey = “4” /label = Ori\-EC\(pMB) ¹⁾coding sequence ²⁾on thecomplementary strand

EXAMPLE 4 Mutagenesis of the C. glutamicum LysC Gene

The site-directed mutagenesis of the C. glutamicum lysC gene (Example 3)was carried out with the QuickChange Kit (Stratagene/USA) following themanufacturer's instructions. The mutagenesis was carried out in theplasmid pCIS lysC, SEQ ID NO:69. The following oligonucleotide primerswere synthesized for the substitution of thr311 for 311ile with the aidof the Quickchange method (Stratagene):

SEQ ID NO:70 5′-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG-3′ SEQ ID NO:715′-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG-3′

The use of these oligonucleotide primers in the Quickchange reactionbrings about a substitution of the nucleotide in position 932 (T for C)in the lysC gene (cf. SEQ ID NO:72) and an amino acid substitution inposition 311 (Thr→Ile) (cf. SEQ ID NO:73) in the corresponding enzyme.The resulting amino acid substitution Thr311Ile in the lysC gene wasconfirmed by sequencing following transformation into E. coli XL1-blueand plasmid preparation. The plasmid was named pCIS lysC thr311ile andis listed as SEQ ID NO:74. The corresponding plasmid map is shown inFIG. 2.

The sequence SEQ ID NO:74 encompasses the following essentialpart-regions:

LOCUS pCIS\lysC\thr311ile  5860 bp  DNA  circular FEATURESLocation/Qualifiers CDS¹⁾   155 . . . 1420 /vntifkey = “4” /label = lysCCDS   complement²⁾(3935 . . . 5356) /vntifkey = “4” /label =sacB\(Bacillus\subtilis) promoter   complement(5357 . . . 5819)/vntifkey = “30” /label = Promotor\sacB C_region   complement(3913 . . .3934) /vntifkey = “2” /label = sacB\downstream region CDS   1974 . . .2765 /vntifkey = “4” /label = Kan\R CDS   complement(3032 . . . 3892)/vntifkey = “4” /label = Ori\-EC\(pMB) ¹⁾coding sequence ²⁾on thecomplementary strand

The plasmid pCIS lysC thr311ile was transformed into C. glutamicumLU1479 by means of electroporation as described by Liebl, et al. (1989)FEMS Microbiology Letters 53:299-303. Modifications of the protocol aredescribed in DE-A-10046870. The chromosomal arrangement of the lysClocus of individual transformants was verified using standard methods bymeans of Southern blotting and hybridization as described in Sambrook etal. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor.It was thus ensured that the transformants were transformants which havethe transformed plasmid integrated at the lysC locus by homologousrecombination. After such colonies were grown overnight in media withoutantibiotic, the cells were plated onto a sucrose CM agar medium (10%sucrose) and incubated for 24 hours at 30° C.

Since the sacB gene which is present in the vector pCIS lysC thr311ileconverts sucrose into a toxic product, only those colonies are capableof growing which have the sacB gene deleted between the wild-type lysCgene and the mutated gene lysC thr311ile by a second homologousrecombination step. During the homologous recombination, either thewild-type gene or the mutated gene can be deleted together with the sacBgene. If the sacB gene is removed together with the wild-type gene, theresult is a mutated transformant.

Growing colonies were picked and examined for a kanamycin-sensitivephenotype. Clones with deleted SacB gene must simultaneously showkanamycin-sensitive growth behavior. Such Kan-sensitive clones wereexamined for their lysine productivity in a shaker flask (see Example6). For comparison, the untreated strain LU1479 was cultured. Cloneswhose lysine production was increased in comparison with the controlwere selected, chromosomal DNA was obtained, and the correspondingregion of the lysC gene was amplified by a PCR reaction and sequenced.Such a clone with the characteristic of an increased lysine synthesisand a confirmed mutation in lysC at position 932 was termed LU1479 lysC311 ile).

EXAMPLE 5 Generation of Ethionine-resistant C. Glutamicum Strains

In the second step of the strain construction, the resulting strainLU1479 lysC 311ile (Example 4) was treated in order to induce ethionineresistance (Kase, H. Nakayama K. Agr. Biol. Chem. 39 153-106 1975L-methionine production by methionine analog-resistant mutants ofCorynebacterium glutamicum). An overnight culture in BHI medium (Difco)was washed in citrate buffer (50 mM pH 5.5) and treated for 20 minutesat 30° C. with N-methylnitrosoguanidine (10 mg/ml in 50 mM citratepH5.5). After treatment with the chemical mutagenN-methyl-nitrosoguanidine, nitrosoguanidine ,the cells were washed(citrate buffer 50 mM pH 5.5) and plated onto a medium composed of thefollowing components, based on 500 ml: 10 g (NH₄)₂SO₄, 0.5 g KH₂PO₄, 0.5g K₂HPO₄, 0.125 g MgSO₄.7H₂O, 21 g MOPS, 50 mg CaCl₂, 15 mgproteocatechuate, 0.5 mg biotin, 1 mg thiamine, 5 g/l D,L-ethionine(Sigma Chemicals Deutschland), pH 7.0. The medium additionally comprised0.5 ml of a microsalt solution of: 10 g/l FeSO₄.7H₂O, 1 g/l MnSO₄*H₂O,0.1 g/l ZnSO₄*7H₂O, 0.02 g/l CuSO_(4,) 0.002 g/l NiCl₂*6H₂O. All saltswere dissolved in 0.1M HCl. The finished medium was filter-sterilizedand, after addition of 40 ml sterile 50% glucose solution, treated withliquid sterile agar in a final concentration of 1.5% agar and pouredinto culture dishes.

Mutagen-treated cells were placed onto plates containing theabove-described medium and incubated for 3-7 days at 30° C. Resultingclones were isolated, isolated individually at least once on theselection medium and then examined for their methionine productivity inmedium II in a shake flask (see Example 6).

EXAMPLE 6 Methionine Production with Strain LU1479 LysC311ile ET-16

The strains produced in Example 5 were grown for 2 days at 30° C. on anagar plate with CM medium.

CM agar:

10.0 g/l D-glucose, 2.5 g/l NaCl, 2.0 g/l urea, 10.0 g/l Bacto peptone(Difco), 5.0 g/l yeast extract (Difco), 5.0 g/l beef extract (Difco),22.0 g/l agar (Difco), autoclaved (20 min., 121° C.)

The cells were subsequently scraped off the plate and resuspended insaline. For the main culture, 10 ml of medium II and 0.5 g autoclavedCaCO₃ (Riedel de Haen) in a 100 ml Erlenmeyer flask were inoculated withthe cell suspension until an OD600 nm of 1.5 was reached and incubatedfor 72 hours at 30° C. on an orbital shaker at 200 rpm.

Medium II:

40 g/l Sucrose 60 g/l Molasses (based on 100% sugar content) 10 g/l(NH₄)₂SO₄ 0.4 g/l MgSO₄*7H₂O 0.6 g/l KH₂PO₄ 0.3 mg/l Thiamine*HCl 1 mg/lBiotin (from a 1 mg/ml filter-sterilized stock solution which had beenbrought to pH 8.0 with NH₄OH) 2 mg/l FeSO₄ 2 mg/l MnSO₄brought to pH 7.8 with NH₄OH, autoclaved (121° C., 20 min). In addition,vitamin B12 (hydroxycobalamine Sigma Chemicals) from a stock solution(200 μg/ml, filter-sterilized) was added to a final concentration of 100μg/l.

The methionine formed, and other amino acids in the culture broth, were[lacuna] with the aid of the amino acid acid determination method fromAgilent using a Agilent 1100 Series LC System HPLC. Derivatizationbefore the column separation with ortho-phthalaldehyde enabled thequantification of the amino acids formed. The amino acid mixture wasseparated on a Hypersil AA column (Agilent).

Those clones whose methionine productivity was at least twice as high asthat of the original strain LU1479 lysC 311ile were isolated. Such aclone was employed for the further experiments and was named LU1479 lysC311ile ET-16.

EXAMPLE 7 Cloning MetY from Mycobacterium tuberculosis and Cloning intothe Plasmid pC Phsdh MetY_Mt

Chromosomal DNA of Mycobacterium tuberculosis was obtained from theAmerican Type Strain Culture Collection (ATCC, Atlanta-USA) from strainATCC 25584. Chromosomal DNA from C. glutamicum ATCC 13032 was preparedby the method described by Tauch et al. (1995) Plasmid 33:168-179 orEikmanns et al. (1994) Microbiology 140:1817-1828.

Using the oligonucleotide primers SEQ ID NO:75 and SEQ ID NO:76, thechromosomal DNA from C. glutamicum as template and Pfu Turbo polymerase(Stratagene), an approx. 180 base pair DNA fragment was amplified fromthe noncoding 5′ region (promoter region) of homoserine dehydrogenase(HsDH) with the aid of the polymerase chain reaction (PCR) followingstandard methods, such as Innis et al. (1990) PCR Protocols. A Guide toMethods and Applications, Academic Press. The amplified fragment isflanked at its 5′ end by a BamHI restriction cleavage site and at its 3′end by a region which is homologous to metY from Mycobacterumtuberculosis and has been introduced via the oligo.

SEQ ID NO:75 5′-GAGAGGATCCGGAAGGTGAATCGAATTTCGG-3′ and SEQ ID NO:765′-CTATTGCTGTCGGCGCTCATGATTCTCCAAAAATAATCGC-3′

The resulting DNA fragment was purified using the GFX™PCR, DNA and gelband purification kit (Amersham Pharmacia, Freiburg) following themanufacturer's instructions.

Starting from the chromosomal DNA from Mycobacterium tuberculosis astemplate for a PCR reaction, metY was amplified with the aid of theGC-rich PCR system (Roche Diagnostics, Mannheim) following themanufacturer's instructions, using the oligonucleotide primers SEQ IDNO:77 and SEQ ID NO:78. The amplified fragment is flanked at its 3′ endby an XbaI restriction cleavage site which has been introduced via theoligo.

SEQ ID NO:77 5′-ATGAGCGCCGACAGCAATAG-3′ and SEQ ID NO:785′-GAACTCTAGATCAGAACGCCGCCACGGAC-3′

The approximately 1.4 kb DNA fragment which was obtained was purifiedwith the GFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) following the manufacturer's instructions.

In a further PCR reaction, the two fragments obtained above wereemployed jointly as template. Owing to the regions which are homologousto the metY fragment and which have been introduced with theoligonucleotide primer SEQ ID NO:76, the two fragments anneal with oneanother during the PCR reaction and are elongated to a continuous DNAstrand by the polymerase employed. The standard method was modifiedinasfar as the oligonucleotide primers used, SEQ ID NO:75 and SEQ IDNO:78, were only added to the reaction at the beginning of the 2ndcycle.

The amplified DNA fragment, which was approximately 1.6 kb in size, waspurified with the GFX™PCR, DNA and gel band purification kit followingthe manufacturer's instructions. Thereafter, it was cleaved with therestriction enzymes BamHI and XbaI (Roche Diagnostics, Mannheim) andseparated by gel electrophoresis. The approximately 1.6 kb DNA fragmentwas subsequently isolated from the agarose using the GFX™PCR, DNA andgel band purification kit (Amersham Pharmacia, Freiburg).

The vector pClik5MCS SEQ ID NO:65, hereinbelow referred to as pC, wascleaved with the restriction enzymes BamHI and XbaI (Roche Diagnostics,Mannheim), and, after separation by electrophoresis, a 5 kb fragment wasisolated with the GFX™PCR, DNA and gel band purification kit.

The vector fragment together with the cleaved and isolated PCR fragmentwere ligated with the aid of the Rapid DNA ligation kit (RocheDiagnostics, Mannheim) following the manufacturer's instructions and theligation reaction was transformed into competent E. coli XL-1Blue(Stratagene, La Jolla, USA) by standard methods as described in Sambrooket al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor,(1989)). Selection for plasmid-bearing cells was achieved by plating onkanamycin (20 μg/ml)—containing LB agar (Lennox, 1955, Virology, 1:190).

The plasmid DNA preparation was carried out by methods of, and usingmaterials from, Quiagen. Sequencing reactions were carried out asdescribed by Sanger et al. (1977) Proceedings of the National Academy ofSciences USA 74:5463-5467. The sequencing reactions were separated andevaluated by means of ABI Prism 377 (PE Applied Biosystems,Weiterstadt).

The resulting plasmid pC Phsdh metY_Mt (Mycobacterium tuberculosis) islisted as SEQ ID NO:79. The corresponding plasmid map is shown in FIG.3.

The sequence SEQ ID NO:79 encompasses the following essentialpart-regions:

LOCUS pC\Phsdh\metY_Mt 6591 bp DNA circular 21-JUL-2003 FEATURESLocation/Qualifiers CDS   156 . . . 1505 /vntifkey = “4” /label =metY\aus\M\tuberculosis CDS   1855 . . . 2646 /vntifkey = “4” /label =Kan\R CDS   4927 . . . 6048 /vntifkey = “4” /label = Rep\Protein CDS  3919 . . . 4593 /vntifkey = “4” /label = ORF\1 CDS   complement(2913 .. . 3773) /vntifkey = “4” /label = Ori\-EC\(pMB)

EXAMPLE 8 Transformation of the Strain LU1479 LysC 311ile ET-16 With thePlasmid pC Phsdh MetY_Mt

The strain LU1479 lysC 311ile ET-16 was transformed with the plasmid pCPhsdh metY_Mt by the above-described method (Liebl, et al. (1989) FEMSMicrobiology Letters 53:299-303). The transformation mixture was platedonto CM plates which additionally comprised 20 mg/l kanamycin in orderto achieve a selection for plasmid-containing cells. Resultingkanamycin-resistant clones were picked and isolated individually. Themethionine productivity of the clones was examined in a shake-flaskexperiment (see Example 6). The strain LU1479 lysC 311ile ET-16 pC PhsdhmetY_Mt produced significantly more methionine in comparison with LU1479lysC 311ile ET-16.

1. A method for the fermentative production of L-methionine, whichcomprises the following steps: a) fermenting in a medium cells of acoryneform bacterium for producing L-methionine, the coryneform bacteriaexpressing at least one heterologous nucleotide sequence which codes fora protein with O-acetylhomoserine sulfhydrolase (metY) activity, whereinthe heterologous nucleotide sequence comprises a nucleotide sequenceencoding a metY protein having an amino acid sequence as set forth inSEQ ID NO: 4 or comprises a nucleotide sequence encoding a metY proteinhaving an amino acid sequence with 95% homology or more to the sequenceas set forth in SEQ ID NO: 4; b) concentrating L-methionine in themedium or in the bacterial cells, and c) isolating L-methionine.
 2. Themethod as claimed in claim 1, wherein the metY-encoding nucleotidesequence comprises a coding sequence as set forth in SEQ ID NO:
 3. 3.The method as claimed in claim 1, wherein the metY-encoding sequencecodes for a protein with metY activity, the protein comprising an aminoacid sequence as set forth in SEQ ID NO:
 4. 4. The method as claimed inclaim 1, wherein the coding metY sequence is a DNA or RNA which can bereplicated in coryneform bacteria or is stably integrated into thechromosome.
 5. The method as claimed in claim 4, wherein the bacteria isa) a bacteria strain transformed with a plasmid vector carrying at leastone copy of the coding metY sequence under the control of regulatorysequences, or b) a strain in which the coding metY sequence has beenintegrated into the bacteria chromosome.
 6. The method as claimed inclaim 1, wherein the coding metY sequence is overexpressed.
 7. Themethod as claimed in claim 1, wherein the bacteria are fermented inwhich additionally at least one further gene of the biosynthetic pathwayof L-metlaionine has been overexpressed.
 8. The method as claimed inclaim 1, wherein the coryneform bacteria are fermented in which, at thesame time, at least one of the genes selected from among a) the genelysC, which encodes an aspartate kinase, b) theglyceraldehyde-3-phosphate dehydrogenase-encoding gene gap, c) the3-phosphoglycerate kinase-encoding gene pgk, d) the pyruvatecarboxylase-encoding gene pyc, e) the triose phosphateisomerase-encoding gene tpi, f) the homoserineO-acetyltransferase-encoding gene metA, g) the cystathioninegamma-synthase-encoding gene metB, h) the cystathioninegamma-lyase-encoding gene metC, i) serinehydroxymethyltransferase-encoding gene glyA, j) the methylenetetrahydrofolate reductase-encoding gene metF, k) the vitamin B12-dependent methionine synthase-encoding gene metH, l) the phophoserineaminotransferase-encoding gene serC, m) the phosphoserinephosphatase-encoding gene serB, n) the serine acetyltransferase-encodinggene cysE, and o) the gene hom, which encodes a homoserinedehydrogenase, is overexpressed.
 9. The method as claimed in claim 1,wherein the coryneform bacteria are fermented in which, at the sametime, at least one of the genes selected from among a) the homoserinekinase-encoding gene thrB, b) the threonine dehydratase-encoding geneilvA, c) the threonine synthase-encoding gene thrC, d) themeso-diaminopimelato D-dchydrogenase-encoding gene ddh, e) thephosphoenolpyruvate carboxykinase-encoding gene pck, f) theglucose-6-phosphate 6-isomerase-encoding gene pgi, g) the pyruvateoxidase-encoding gene poxB, h) the dihydrodipicolinate synthase-encodinggene dapA, i) the dihydrodipicolinate reductase-encoding gene dapB; andj) the diaminopicolinate decarboxylase-encoding gene, is attenuated bychanging the rate of expression.
 10. The method as claimed in claim 1,wherein the coryneform bacterium is of the species Corynebacteriumglutamicum.
 11. A method for producing an L-methionine-containing animalfeed additive from fermentation broths, which comprises the followingsteps: a) culturing and fermenting L-methionine-producing cells of acoryneform bacterium in a fermentation medium; b) removing water fromthe L-methionine-containing fermentation broth; c) removing from 0 to100% by weight of the biomass formed during fermentation; and d) dryingthe fermentation broth obtained according to b) and/or c), in order toobtain the animal feed additive in the desired powder or granule form;wherein the coryneform bacteria express at least one heterologousnucleotide sequence which codes for a protein with O-acetylhomoserinesulfhdrolase (metY) activity, where the heterologous nucleotide sequencecomprises a nucleotide sequence encoding a metY protein having an aminoacid sequence as set forth in SEQ ID NO: 4 or comprises a nucleotidesequence encoding a metY protein having an amino acid sequence with 95%homology or more to the sequence as set forth in SEQ ID NO:
 4. 12. Themethod of claim 1, wherein the metY-encoding sequence is derived fromMycobacterium tuberculosis.
 13. The method of claim 1, wherein thecoryneform bacteria are fermented in which, at the same time, a genelysC derived from a coryneform bacteria, which encodes an aspartatekinase, is overexpressed.
 14. The method of claim 13, wherein the lysCgene is derived from Corynebacterium glutamicum.
 15. A method for theproduction of L-methionine, which comprises the following steps: a)fermenting in a medium cells of a coryneform bacterium for producing ofL-methionine, the coryneform bacteria expressing at least oneheterologous nucleotide sequence which codes for a protein withO-acetylhomoserine sulfhydrolase (metY) activity, wherein theheterologous nucleotide sequence comprises a nucleotide sequence having95% identity or more to the sequence as set forth in SEQ ID NO: 3; b)concentrating L-methionine in the medium or in the bacterial cells; andc) isolating L-metbionine.
 16. The method of claim 15, wherein thecoding metY sequence is a DNA or RNA which can be replicated incoryneform bacteria or is stably integrated into the chromosome.
 17. Themethod of claim 15, wherein a) a bacteria strain transformed with aplasmid vector carrying at least one copy of the coding metY sequenceunder the control of regulatory sequences is used, or b) a strain inwhich the coding metY sequence has been integrated into the bacteriachromosome is used.
 18. The method of claim 15, wherein the coding metYsequence is overexpressed.
 19. The method of claim 15, wherein thecoryneform bacterium is of the species Corynebacterium glutamicum. 20.The method of claim 15, wherein bacteria are fermented in whichadditionally at least one further gene of the biosynthetic pathway ofL-methionine is overexpressed.
 21. The method of claim 20, wherein theat least one further gene is a gene lysC derived from a coryneformbacteria, which encodes an aspartate kinase.